This invention relates to a method and apparatus for automatic measurement of fluorescence and more particularly to an analytical method and apparatus applicable to measurement of a fluorescent substance created by adding a latently fluorescent reagent to a sample to be tested.
In the past, a radio-immunoassay method using radioisotopes has been employed widely in order to measure, with immunologic methods, a small amount of substances in a biological sample, but recently a measurement method using an enzyme or a liposome has been predominant.
An enzyme immunoassay method is disclosed in, for example, JP-A-62-50662. In this example, a complex of an antigen, an antibody and an enzyme is created on the surface of a bead and the intensity of fluorescence caused by an enzyme reaction, which develops when a substrate is brought into contact with the complex, is measured.
The above JP-A-62-50662 uses an ordinary fluorophotometer to measure reaction solutions and fails to refer to an apparatus using an automated reaction system and a fluorophotometer in combination.
On the other hand, in the field of automatic chemical analyzers, the analyzer itself changes (drifts) with time so that measured values are affected, by such a drift change, and therefore countermeasures against this problem have been contrived. For example, U.S. Pat. No. 4,043,756 discloses a method of photometry of light which is absorbed by reaction solutions of samples to be tested which are drawn into flow cells. Before carrying out measurements of the test samples, a reaction solution of a reference sample and a reagent blank are first measured with a view of correcting a standard curved, and a drift variation of the analyzer per se is corrected using a corrected standard curve.
When designing an immunoanalyzer by incorporating a fluorophotometer into an automatic analyzer, it is of significance to ensure high stability of the detection system and high reliability of results of measurement in the immunoanalyzer. In the measurement, quantitative measurement of a small amount of antigens or antibodies in a biological sample is needed and abnormality in performance of the optical system for fluorophotometry leads to impairment of analysis of small amounts. Further, degradation of the reagent is a factor in increasing measurement error in the analysis of small amounts.
The aforementioned U.S. Pat. No. 4,043,756 describes drift variations of the analyzer but does not consider the performance degradation of the optical system and the reagent degradation. A reference sample used in this literature is a reaction solution prepared by reacting the same element as a target element in a test sample in the same way. In immunoanalysis, however, it is difficult to obtain a chemically stable solution of a substance which may be used as a reference sample in reactions.